A genetic framework for regulation and seasonal adaptation of shoot architecture in hybrid aspen

Jay P. Mauryaa,b, Pal C. Miskolczia, Sanatkumar Mishraa, Rajesh Kumar Singha, and Rishikesh P. Bhaleraoa,1

aUmeå Science Centre, Department of Forest Genetics and , Swedish University of Agricultural Sciences, SE-901 87 Umeå, Sweden; and bDepartment of , Institute of Science, Banaras Hindu University, Varanasi 221005, Uttar Pradesh, India

Edited by Ronald R. Sederoff, North Carolina State University, Raleigh, NC, and approved April 14, 2020 (received for review March 14, 2020) Shoot architecture is critical for optimizing plant adaptation and time–related factor RAV1 has been validated in productivity. In contrast with annuals, branching in perennials branching in (17–19). However, information on branching native to temperate and boreal regions must be coordinated with control in perennials is fragmented, and there is a significant gap seasonal growth cycles. How branching is coordinated with in our knowledge of how branching is controlled and integrated seasonal growth is poorly understood. We identified key compo- with seasonal growth cycles in perennial trees (20). Therefore, nents of the genetic network that controls branching and its using functional genetic approaches, we elucidated the genetic regulation by seasonal cues in the model hybrid aspen. network that mediates control of branching and its regulation by Our results demonstrate that branching and its control by sea- seasonal cues in the model tree hybrid aspen. These studies re- sonal cues is mediated by mutually antagonistic action of aspen veal the key role played by the mutually antagonistic action of orthologs of the flowering regulators TERMINAL 1 LAP1 and TFL1 in mediating control of branching and its re- (TFL1)andAPETALA1 (LIKE APETALA 1/LAP1). LAP1 promotes sponses to hormonal and seasonal cues. Our results show that branching through local action in axillary . LAP1 acts in a components involved in photoperiodic control of seasonal -dependent manner, stimulating expression of the - growth have been recruited along with tree orthologs of pre- AIL1 BRANCHED1 cycle regulator and suppressing expression to viously described branching regulators to control of branching promote branching. Short photoperiod and low temperature, the and its adaptation to seasonal changes. major seasonal cues heralding winter, suppress branching by si- multaneous activation of TFL1 and repression of the LAP1 path- Results DEVELOPMENTAL BIOLOGY way. Our results thus reveal the genetic network mediating Dormancy- and Activation-Related Genes Are Differentially control of branching and its regulation by environmental cues fa- Expressed during Axillary Bud Outgrowth. To identify how shoot cilitating integration of branching with seasonal growth control in branching is regulated in trees, we monitored the temporal ex- perennial trees. pression of genes in axillary buds of hybrid aspen (P. tremula × tremuloides) clone T89 before and after decapitation of shoot shoot architecture | branching | seasonal growth | perennial trees | apex (21) (SI Appendix, Fig. S1). We selected genes previously hybrid aspen implicated in bud dormancy and bud break (both apical and axillary buds). The first visible signals of activity in the buds were hoot architecture has a central role in adaptation and opti- detected 24 to 48 h after decapitation, as buds appeared to grow Smizing plant productivity. Hence the outgrowth of axillary and separate from the stem. Transcript levels of genes previously buds is tightly regulated in response to endogenous signals and implicated in bud outgrowth, such as BRANCHED1/TEOSINTE exogenous environmental cues. Branching has been extensively Arabidopsis studied in the model and pea. These studies Significance have shown tight regulation of axillary bud outgrowth, revealed the key genetic components controlling branching, and identified Control of branching is critical for optimizing growth and ad- roles played by hormonal and signals (1–3). The plant aptation in plants. In contrast with annuals, in perennial plants and indole-acetic acid (IAA/) sup- growing in temperate and boreal regions, branching must be press branching, whereas cytokinin and such as nitrogen controlled temporally to adapt to seasonal changes. The mo- and sucrose promote it (4–13). Hormonal and nutrient signaling lecular pathways regulating branching and its adaptation to pathways converge on BRANCHED1 (BRC1) seasonal changes are not well understood in perennial plants. (2, 14), which integrates the various inputs and mediates control of ’ We identified the genetic network underlying the control of branching by regulating axillary buds potential to grow. branching and elucidated its role in branching by seasonal In contrast with annuals, perennials, particularly of boreal and cues in model tree hybrid aspen. Our results reveal compo- temperate regions, face extreme annual variation in temperature nents mediating photoperiodic control of growth in trees, and day length. Essential adaptations of perennials to these and conserved branching regulators have been integrated seasonal changes include synchronized cycles of growth and into a genetic network to control branching and facilitate its dormancy. Prior to winter, shoot growth is arrested and shoot adaptation to seasonal changes experienced by long-lived apical and arrested primordia are enclosed within perennial plants. an apical bud, and precocious activation of shoot growth is prevented by the establishment of dormancy (15, 16). Like shoot Author contributions: J.P.M. and R.P.B. designed research; J.P.M., P.C.M., S.M., and R.K.S. growth, branching needs to be tightly controlled to adapt to performed research; R.P.B. contributed new reagents/analytic tools; J.P.M. and R.P.B. seasonal changes. Inadvertent activation of axillary bud out- analyzed data; and J.P.M. and R.P.B. wrote the paper. growth, late in the season, would cause fatal damage in newly The authors declare no competing interest. formed shoots from early winter frost. This article is a PNAS Direct Submission. Whereas branching is well studied in annual models like Published under the PNAS license. Arabidopsis and pea, much less is known about control of 1To whom correspondence may be addressed. Email: [email protected]. branching in perennial trees. Changes in gene expression in ax- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ illary buds have been described, and the role of a few compo- doi:10.1073/pnas.2004705117/-/DCSupplemental. nents including , BRC1 orthologs, and flowering-

www.pnas.org/cgi/doi/10.1073/pnas.2004705117 PNAS Latest Articles | 1of8 Downloaded by guest on September 26, 2021 BRANCHED1, CYCLOIDEA, PCF 18 (BRC1/TCP18, a homolog the branching phenotype (SI Appendix, Fig. S5). In contrast with of Arabidopsis BRANCHED 1)(22,23),andTERMINAL FLOWER FT overexpression, LAP1 overexpression led to a significant in- 1/CENTRORADIALIS 1 (TFL1/CEN1) (24) were down-regulated. crease in branching with outgrowth from nearly all of the axillary In contrast, transcript levels of AINTEGUMENTA-LIKE 1 (AIL1), buds (Fig. 2 A and B). a known cell proliferation regulator (25–27), and Arabidopsis The up-regulation of LAP1 expression in TFL1-RNAi axillary thaliana HISTIDINE PHOSPHOTRANSMITTER 4 (AHP4, puta- buds and the LAP1oe phenotype suggested that TFL1 sup- tively involved in cytokinin signaling) (28) were up-regulated 8 to presses axillary bud outgrowth by suppressing LAP1 expression. 24 h after shoot apex decapitation before any visible change in To test this hypothesis, we generated TFL1-RNAi aspen plants SI Appendix A–D axillary buds ( ,Fig.S2 ). Thus, genes implicated in with LAP1 activity knocked out by clustered regularly inter- bud dormancy and outgrowth display dynamic changes in expres- spaced short palindromic repeats (CRISPR)-Cas9 (Fig. 2C and sion preceding and during axillary bud outgrowth. SI Appendix, Fig. S6). The resulting TFL1-RNAi/LAP1ko plants TFL1/CEN1 resembled WT producing extremely few branches, in contrast Is a Branching Repressor. The correlation between down- LAP1 regulation of TFL1 expression preceding axillary bud outgrowth with TFL1-RNAi plants. Thus, loss of function of could and its proposed role as a negative regulator of apical bud break suppress increased branching resulting from down-regulation of TFL1 LAP1 (SI Appendix, Fig. S2B) (24) prompted us to investigate its role in . The enhanced expression of in TFL1-RNAi and axillary bud outgrowth. RNA interference (RNAi)-mediated suppression of branching in TFL1-RNAi by loss of function of down-regulation of TFL1 (SI Appendix, Fig. S3) resulted in more LAP1 indicate that LAP1 is a downstream target in TFL1- branching than in wild-type (WT) plants (Fig. 1A). Moreover, mediated control of branching. upon decapitation, TFL-down-regulated (TFL1-RNAi) and -overexpressing (TFL1oe) lines had significantly higher and LAP1 Acts Locally in Promoting Branching by Modulating Key lower frequencies of axillary bud outgrowth, respectively, than Branching-Related Genes. Following identification of the role of WT plants (SI Appendix, Fig. S4 A and B). These results suggest LAP1 in branching, we investigated if LAP1 acts locally or sys- that TFL1 acts to repress branching in hybrid aspen. temically in promoting branching because some key regulators, e.g., strigolactones, act as long-range signals in branching (8, 9, LAP1 Is the Downstream Target in TFL1-Mediated Suppression of 39–42). We self-grafted wild type (WT/WT), and LAP1oe (LAP1oe/ Branching. Next, we investigated the downstream targets of LAP1oe) and wild-type and LAP1oe scions were grafted onto TFL1 involved in control of axillary bud outgrowth. TFL1 is LAP1oe (WT/LAP1oe) and wild-type (LAP1oe/WT) stocks, known to act antagonistically to the flowering regulator respectively, and axillary bud outgrowth was monitored in the FLOWERING LOCUS T FT APETALA 1 AP1 ( ) and ( ) during scions after . The wild-type scions in WT/WT grafts did – FT AP1 flower development (29 32). Both and orthologs par- not make any branches whereas LAP1oe scions in LAP1oe/ ticipate in seasonal regulation of shoot apex activity, as well of LAP1oe grafts continued to make branches both in scion and bud break (33–35). Also, both rice and cotton orthologs of FT root stocks (SI Appendix, Fig. S7 A and B). While the LAP1 root promote shoot branching (36, 37). Therefore, we investigated the stocks did not induce branches in WT scions, the LAP1oe scions expression of hybrid aspen FT2 and LAP1 in the axillary buds of FT2 LAP1 that were grafted onto WT root stocks continued to produce WT and TFL1-RNAi hybrid poplar plants. Both and LAP1 were expressed in axillary buds more strongly in TFL1-RNAi branches. These results suggest that acts locally in promoting plants than in WT plants (Fig. 1 B and C). branching. The enhanced expression of FT and LAP1 in TFL1-RNAi We then monitored gene expression in axillary buds of LAP1oe LAP1 axillary buds and the role of FT orthologs in branching promp- to identify downstream targets in the pathway and elucidate LAP1 BRC1 TFL1 ted us to ask if FT or LAP1 can promote shoot branching in how promotes branching. Expression of and , trees. In hybrid aspen, two closely related FT orthologs (FT1 and which both act negatively in branching, was significantly weaker in FT2) have been described so far (38). Hence, we investigated the LAP1oe axillary buds than in the WT buds (Fig. 2 D and E). In whether overexpression of FT1 or FT2 enhances branching, as contrast, expression of AIL1 and AHP4, a cytokinin signaling- observed upon down-regulation of TFL1. Neither FT1 nor FT2 related gene, were significantly up-regulated in axillary buds of overexpressor lines differed significantly from the wild type in LAP1oe compared to WT buds (Fig. 2 F and G).

Fig. 1. TFL1 is a repressor of branching. (A) Numbers of branches formed by WT and TFL1-RNAi plants in LD conditions. (B and C) Expression levels of FT2 and LAP1 in WT and TFL1-RNAi plants’ axillary buds in long days, respectively. Error bars indicate SEM. The expression values are normalized to the reference gene UBQ and averages of three biological replicates. Statistical analysis was done using unpaired t test. Asterisks (*) and (**) indicate significant differences from WT at P ≤ 0.001 and 0.0001, respectively.

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Fig. 2. LAP1 promotes branching. (A) Branching phenotypes of WT and LAP1oe plants grown in LD conditions. (B) Numbers of axillary branches formed by plants illustrated in A.(C) Numbers of branches formed by WT, TFL1-RNAi, and LAP1ko/TFL1-RNAi lines in LD conditions. (D–G) Real-time PCR data showing relative transcript levels of BRC1, TFL1, AHP4, and AIL1 in axillary buds of WT and LAP1oe plants grown under LD conditions, respectively. Expression values are normalized to the reference gene UBQ and averages of three biological replicates. Error bars indicate SEM. Statistical analysis was done using unpaired t test. Asterisks (*) and (**) indicate significant differences from WT at P ≤ 0.001 and 0.01, respectively.

LAP1 Promoted Branching Is Mediated Via Suppression of BRC1. In However, in hybrid aspen four nearly identical AIL genes act re- several plants, including poplar, BRC1 and its homolog BRC2 act dundantly (27). To overcome this genetic redundancy, we generated as branching repressors (18, 22). The reduced expression of a dominant repressor, antagonizing the function of endoge- BRC1 in LAP1oe plants and recent data showing that LAP1 can nous AIL genes by expressing a fusion of AIL1 with an SRDX bind to the promoter of BRC1 (43) suggest that LAP1 could domain (a synthetic repressor) (44). We expressed the AIL1-SRDX promote branching by suppressing BRC1 expression in axillary repressor in the LAP1oe background (SI Appendix,Fig.S9A). buds. To address this hypothesis, we expressed BRC1 in the The resulting AIL1-SRDX/LAP1oe plants produced signifi- LAP1oe background (SI Appendix, Fig. S8A) and investigated cantly fewer branches than the parental LAP1oe plants (Fig. the resulting branching phenotype. BRC1oe/LAP1oe double 3B), thus confirming that LAP1 acts via AIL genes in promoting transgenic lines produced significantly fewer branches than branching. LAP1oe plants (SI Appendix, Fig. S8 B and C), indicating that BRC1 is a downstream target of LAP1 and that LAP1 promotes LAP1 Pathway Interacts with Cytokinin Signaling in Branching. Plant branching by repressing BRC1 expression in hybrid aspen. cytokinin promotes shoot branching. Therefore, we investigated whether are involved in LAP1-mediated LAP1 Requires AIL1 Activity to Promote Branching. The expression promotion of branching. For this, we generated transgenic hybrid of AIL1 transcription factor is up-regulated in axillary buds after aspen plants overexpressing LAP1 in a BHK4-CKX2 background decapitation as well in LAP1oe (Fig. 2F). AIL1 transcription (LAP1oe/BHK4-CKX2) (SI Appendix, Fig. S9B), which had re- factor is a downstream target of LAP1 in photoperiodic control duced cytokinin levels due to overexpression of Arabidopsis of growth (34). Ectopic expression of AIL1 (AIL1oe) resulted in CYTOKININ OXIDASE 2 (45). In contrast with LAP1oe, LAP1oe/ more branching than in WT plants (Fig. 3A), suggesting that BHK4-CKX2 plants resembled WT plants and displayed nearly AIL1 could act in LAP1-mediated promotion of branching. To complete suppression of branching (Fig. 3C), indicating that the test this possibility, we suppressed AIL in the LAP1oe background. cytokinin pathway is required for LAP1-mediated promotion of

Maurya et al. PNAS Latest Articles | 3of8 Downloaded by guest on September 26, 2021 Fig. 3. LAP1 acts via AIL and cytokinin to promote branching. Bar graphs show numbers of branches formed by (A) WT and AIL1oe plants; (B) WT, LAP1oe, and AIL1srdx (in LAP1oe background) plants; (C) WT, LAP1oe, and LAP1oe (in BHK4-CKX2 background) plants; and (D) WT, LAP1oe, and AHP4-RNAi (in LAP1oe background) plants grown in LD conditions. Error bars indicate SEM.

branching. This result was corroborated by the phenotype of plants continued to grow in SDs as expected (34) or in low transgenic hybrid aspen plants in which expression of AHP4,a temperature (SI Appendix, Fig. S11), branching was completely putative cytokinin pathway-related gene (28), was down-regulated suppressed in these plants by exposure to SDs or low tempera- in LAP1oe background. As described for LAP1oe/BHK4-CKX2, ture (Fig. 4A and SI Appendix, Fig. S12A). Thus, the seasonal down-regulation of AHP4 expression in a LAP1oe background cues contribute to the control of branching and can override (AHP4-RNAi/LAP1oe) (SI Appendix,Fig.S9C and D)resultedin LAP1-mediated promotion of branching. significantly less branching than in LAP1oe plants (Fig. 3D). Thus, To better understand how seasonal cues control branching, we our data suggest that the promotion of branching by LAP1 re- investigated the expression of genes implicated in bud dormancy quires cytokinin and AHP4. and outgrowth in axillary buds of LAP1oe plants grown in short days or low temperature (Fig. 4 B–E and SI Appendix, Fig. Short Days and Low Temperature Suppress Branching in LAP1oe S12 B–E). The transcript levels of BRC1 and TFL1, which we Plants. In contrast with annual plants, perennial trees need to have shown suppress branching, were up-regulated upon exposure prevent inadvertent activation of branching late in the season as to SDs or low temperature. Conversely, expression of genes such winter approaches to reduce the risk to new shoots from winter as AIL1 and AHP4 that promote branching were down-regulated damage. Short days and non-chilling low temperature (here after in axillary buds of LAP1oe plants grown in SDs or low tempera- referred to as low temperature) are major seasonal cues her- ture (Fig. 4 B–E and SI Appendix,Fig.S12B–E). Thus, seasonal alding autumnal transition to winter and mediate seasonal cues act through known mediators of branching and negate the growth cessation in shoot apex (33–35). To address the poorly effect of LAP1 by activating the expression of negative regulators understood mechanisms whereby these autumnal transition cues and suppressing that of promoters of branching. control branching, we first decapitated wild-type T89 plants grown in short days or low temperature and analyzed their bud out- Suppression of LAP1 Promoted Branching by Seasonal Cues Is growth (SI Appendix,Fig.S10A and B). Both short days (SDs) or Dependent on TFL1. TFL1 is a negative regulator of branching low temperature completely suppressed bud outgrowth following (Fig. 1), and its expression is up-regulated by SDs or low tem- decapitation in contrast with long days. Thus, environmental cues perature in LAP1oe plants (Fig. 4C), indicating that TFL1 up- mediating seasonal control of shoot growth also act on axillary bud regulation could be required for suppression of branching by outgrowth, thereby coordinating apical growth and branching and seasonal cues. Therefore, we examined axillary bud outgrowth in facilitating adaptation to seasonal change. transgenic hybrid aspen in which we down-regulated TFL1 ex- We next examined the molecular basis of seasonal control of pression in LAP1oe (LAP1oe/TFL1-RNAi) (SI Appendix, Fig. axillary bud outgrowth. Because our data indicated that control S13) following exposure to SDs or low temperature. In contrast of shoot growth and branching involves shared components such with LAP1oe, SDs or low temperature could no longer suppress as LAP1, we investigated whether seasonal cues mediating axillary bud outgrowth in LAP1oe/TFL1-RNAi (Fig. 4F and SI branching control act through these components. To address this Appendix, Fig. S12F). These results suggest that suppression of possibility, we investigated branching in LAP1oe plants after branching by seasonal cues in LAP1oe is dependent on TFL1 exposure to SDs or low temperature. While apices of LAP1oe up-regulation.

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Fig. 4. Photoperiodic control of the shoot growth and branching.(A) Branching phenotype of WT and LAP1oe plants grown in SD conditions after leaf tagging (yellow tags). (B–E) Relative transcript levels of indicated bud growth-related genes in axillary buds of LAP1oe plants grown under LD and SD conditions. (F) Numbers of branches produced by WT, TFL1-RNAi, LAP1oe, and LAP1oe/TFL1-RNAi background plants grown in SD conditions. The expression values are normalized to the reference gene UBQ and averages of three biological replicates. Error bars indicate SEM. Statistical analysis was done using unpaired t test. Asterisks (*), (**), and (***) indicate significant differences from WT at P ≤ 0.01, 0.001, and 0.0001, respectively.

Discussion and AHP4, a signaling component implicated in the cytokinin Tight regulation of shoot architecture by controlling the activity pathway, as mediators of branching in hybrid aspen. AIL1 me- of axillary buds is crucial for plants (1, 46, 47). In contrast with diates in photoperiodic control of seasonal growth, whereas cy- annuals such as Arabidopsis and pea, much less is known about tokinin signaling plays a key role in branching (6, 10, 27). Thus, branching control in perennials. Moreover, neither of these an- while gene expression programs in axillary bud outgrowth are nual models display seasonal growth cycles, and how branching conserved across annual and perennial trees, they also suggest in perennials is coordinated with seasonal growth cycles remains that regulators of seasonal growth could be involved in branching poorly understood. To address this knowledge gap, we first in perennials. identified genetic pathways involved in branching and explored Although gene expression studies implicate TFL1 in branch- their role in mediating the control of branching by seasonal cues ing, functional evidence for TFL1 involvement in branching has in hybrid aspen. been lacking in trees. Our data showing increased branching in First, we analyzed changes in expression of key marker genes TFL1-RNAi plants suggest that TFL1 acts as a suppressor of associated with activation of axillary bud outgrowth following branching in hybrid aspen (Fig. 1A). TFL1 has been extensively decapitation in hybrid aspen (SI Appendix, Fig. S2). In agreement studied in the control of flowering, where it acts antagonistically with previous studies (18, 22, 37, 48–50), our data suggest that to the flowering promoters FT and AP1 (29, 30, 31, 32), and we BRC1 down-regulation is an important early event and support observe this in axillary buds (Fig. 1 B and C). Therefore, we its key role as an integrator of various input signals in branching investigated whether the branching mechanism involves a bal- regulation (2, 14). Down-regulation of BRC1 putatively enhances ance between FT and TFL1 as shown in control of floral tran- buds’ potential to grow out (51), and data obtained from both sition in Arabidopsis (52–54). Whereas FT overexpression results gene expression and transgenic analyses with Populus × can- in increased branching in rice and tobacco (36, 37), our results escens clone INRA 717–1B4 indicate the conservation of BRC1 show that LAP1 rather than FT promotes increased branching in function in branching in trees (17, 18). However, gene expression hybrid aspen (Fig. 2 A–C and SI Appendix, Fig. S5). Thus, FT- also identified potential candidates such as TFL1,anegativeregu- TFL1 balance does not appear to be a major determinant of lator of spring bud break; AIL1, a promoter of cell proliferation; branching; instead, TFL1 acts by suppressing expression of LAP1

Maurya et al. PNAS Latest Articles | 5of8 Downloaded by guest on September 26, 2021 in branching regulation in hybrid aspen trees in contrast with (34, 35), has also been recruited for mediating branching and its flowering-time regulation. control by seasonal cues. The dual use of this regulatory module in Whereas systemic signals play important roles in branching (8, seasonal growth at the apex and branching could indicate a simple 9), grafting suggests that LAP1 acts locally in promoting axillary mechanism to coordinate seasonal regulation of these two de- bud outgrowth (SI Appendix, Fig. S7). Gene expression and en- velopmental processes by common cues such as photoperiod or hanced branching in LAP1oe and TFL1RNAi plants indicate low temperature that act as major environmental inputs for au- TFL1 that LAP1 could promote branching by suppressing ex- tumnal transition in boreal and temperate regions. Our results pression. In flowering-time regulation, TFL1 has been shown to AP1 also highlight a striking feature of branching in hybrid aspen. repress , and vice versa (30, 32, 55, 56). The conservation of Whereas plays a key role in repressing axillary this TFL1-AP1 negative loop in axillary buds and the similar bud outgrowth in annuals (as well perennials) (1, 47), seasonal phenotypes of TFL1 down-regulation and LAP1 overexpression cues like SDs or low temperature (that mimic autumn transition) suggest that relative levels of these two mutually antagonistic factors are important for branching. Thus, regulatory features of repress axillary bud outgrowth even when repression via apical the flowering-time pathway appear to have been recruited to dominance is removed in hybrid aspen. We suggest that repression branching regulation in perennial trees. However, rather than of axillary bud outgrowth by seasonal cues plays a protective FT/TFL1 balance, the antagonistic loop between TFL1 and function and that this mechanism may have evolved in perennials LAP1 is important in branching in hybrid aspen. to presumably prevent inappropriate activation of axillary bud In addition to TFL1, LAP1 promotes branching via suppres- outgrowth late in the season even when the shoot apex may be sion of BRC1, the conserved integrator of diverse signaling damaged. pathways in branching regulation in axillary buds (2, 14). In summary, the genetic framework underlying branching Moreover, our data show that LAP1 promotes branching by control in perennials such as trees has incorporated conserved positively regulating AIL1, a positive regulator of the expression components identified in annuals and merged these with ele- of cell-cycle–related genes, such as D-type cyclin genes (34). ments mediating seasonal growth to facilitate coordination of Thus, the LAP1-dependent regulatory module mediating sea- branching with seasonal growth cycles. Despite evident conser- sonal control of growth is conserved in axillary buds. However, vation, there are also notable differences from annuals such as LAP1-promoted branching is also dependent on the cytokinin the key role of the mutually antagonistic TFL1-LAP1 loop and pathway that is known to promote branching (5, 6, 10). Earlier a the regulatory complexity that results in seasonal cues operating AP1 connection between and the cytokinin pathway has been to prevent branching even when the shoot apex is removed. noted in establishment of determinate floral meristems via reg- Moreover, while chilling temperatures promote dormancy re- ulation of cytokinin levels (57) as well as in compound leaf de- lease, our results reveal that nonchilling low temperatures pre- velopment in (58). Thus, the AP1-cytokinin module vent axillary bud outgrowth and the underlying components. appears to be a widely utilized regulatory module across species Thus, our results extend our knowledge of branching regulation to control a wide variety of developmental processes including Arabidopsis branching in trees. Altogether, our data show that regulatory derived primarily from annual models such as and interaction between the AP1 ortholog LAP1 with hormonal pea and reveal how coordination between growth in the shoot pathways and mediators of seasonal growth have been recruited to the branching pathway in addition to their role in seasonal control of growth in trees and flowering in Arabidopsis. Following the identification of genetic components mediating the control of branching, we next investigated how branching is regulated by seasonal cues. In contrast with annuals, perennials like long-lived trees display seasonally synchronized growth cy- cles to adapt to seasonal changes. In these plants, inadvertent activation of axillary bud outgrowth needs to be prevented, e.g., late in the autumn when the risk of damage from chilling injuries increases due to sudden frosts. Whereas chilling-mediated re- lease of bud dormancy is well studied (15, 59), the mechanism underlying seasonal control of branching by autumnal transition cues, such as nonchilling low temperature or SDs, remains poorly understood in trees. Our data indicate that these two major cues, nonchilling low temperature or SDs, that herald autumnal transition to winter and control seasonal growth in trees (15) also play an important role in seasonal control of branching in trees. Both of these cues suppress axillary bud outgrowth and our data suggest that TFL1 is required for suppression of branching by these seasonal cues. However, it is worth noting that axillary bud outgrowth under autumn-mimicking conditions is stringently controlled. SDs and nonchilling low temperature can prevent axillary bud outgrowth even when the shoot apex is removed, and bud outgrowth under these conditions requires simultaneous down-regulation of TFL1, a suppressor, and activation of LAP1. We propose a model (Fig. 5) for genetic control of branching in trees and its regulation by seasonal cues. The signaling path- way mediating control of branching in trees utilizes evolution- BRC1 Fig. 5. Schematic model for branching in trees. Under LD conditions, TFL1 arily conserved components, including and hormones such suppresses LAP1. LAP1 promotes branching by increasing the cytokinin sig- as strigolactones and cytokinins. However, in perennials, where naling and repressing TFL1 and BRC1 expression. Exposure to SDs or low seasonal adaptation is critical for survival, the TFL1-LAP1 temperature (mimicking seasonal transition to winter), TFL1 and BRC1 ex- module, which plays a crucial role in the photoperiodic pathway pression is up-regulated resulting in suppression of branching.

6of8 | www.pnas.org/cgi/doi/10.1073/pnas.2004705117 Maurya et al. Downloaded by guest on September 26, 2021 apex and branching is achieved to adapt to seasonal changes at CKX2 constructs were generated by cloning Arabidopsis CYTOKININ OXIDASE the molecular level in perennial trees. 2 (AtCKX2) along with BIRCH 4 (BHK4) promoter into pGEM-T Easy vector and then transferred to pHTT650 binary vector (45). Materials and Methods All these constructs (pH2GW7-BRC1, pH2GW7-LAP1, BHK4-AtCKX2, pH2GW7- Plant Materials, Growth Conditions, and Tissue Sampling. WT hybrid aspen AHP4-RNAi, pH2GW7-AIL1-SRDX, and pHSE401-LAP1-CRISPR)wereintro- (Populus tremula × tremuloides, clone T89) and transgenic plants were duced into strain GV3101pmp90RK and then used to trans- grown on half-strength Murashige–Skoog medium (Duchefa) under sterile form WT, LAP1oe, and TFL1-RNAi plants to generate single and double conditions for 4 wk and then transferred to soil and cultivated for 4 to 5 wk transgenic lines. All of the primers used for cloning are listed in SI Appendix, in a greenhouse [providing 18 h day/6 h night at 22/18 °C long-day (LD) Table S1. conditions]. Plants were fertilized weekly when growing in the greenhouse. Furthermore, plants were transferred and grown in controlled chambers Generation of Transgenic Hybrid Aspen. All of the transformations were under long-day (18 h day/6 h night at 20/15 °C) or short-day (8 h day/16 h performed as described by Tylewicz et al. (62). night at 20/15 °C) or at a constant temperature of 12 °C (serving as low temperature in long days) for 5 to 6 wk, and then their branches were Grafting of Hybrid Aspen Plants. WT and LAP1oe plants used in the grafting counted. To enhance the visibility of branches, pictures were taken after experiments were grown for 5 wk in a greenhouse providing 18 h light/6 h removing using a Canon EOS digital camera. Tissue samples for gene dark cycles at 22 °C and 60% relative humidity) and then grafted as pre- expression analyses were collected from axillary buds at time points (in- viously reported (45). After 2 wk in long-day conditions, the growing grafts dicated in the figures), immediately frozen in liquid nitrogen, and stored were transferred to a long-day growth chamber (providing 18 h light/6 h at −80 °C. dark cycles at 20/15 °C, 80% relative humidity) and monitored for branch development. Plasmid Constructions and Generation of Transgenic Lines. The generation of LAP1oe, FT1oe, FT2oe, TFL1oe, AIL1oe, and TFL1-RNAi plants has been RNA Isolation and qRT-PCR Analysis. Total RNA was extracted using a Spectrum previously described (24, 27, 34, 35). To generate a BRC1 construct, the Plant Total RNA Kit (Sigma-Aldrich). Ten-microgram portions of RNA were coding sequence (CDS) was amplified by PCR and cloned into pENTR/D-TOPO treated with RNase-free DNase I (Life Technologies, Ambion), and 1-μg donor vector (Invitrogen), sequenced, and subsequently cloned into the portions were then utilized for complementary DNA (cDNA) synthesis us- pH2GW7 plant transformation vector (60) to generate the plasmid pH2GW7- ing an iScript cDNA Synthesis Kit (Bio-Rad). In all experiments, ubiquitin was BRC1. To make AHP4-RNAi constructs, a 265-bp fragment was amplified by used as a reference gene. qRT-PCR experiments were conducted using PCR from a full-length AHP4 CDS template and cloned first into donor vector LightCycler 480 SYBR Green I Master mix and a LightCycler 480 II instrument pENTR/D-TOPO and then into pK7GWIWG2 (I) by LR (attL and attR) reaction (both supplied by Roche). The Δ-cq method was used to calculate the rela- to generate a pH7GWIWG2 (I)-AHP4-RNAi construct. tive expression values of the genes of interest. Primer sequences used in this To generate a TFL1-RNAi/LAP1ko line, we used a CRISPR-Cas9 strategy. An study are listed in SI Appendix, Table S1. DEVELOPMENTAL BIOLOGY LAP1-CRISPR construct was generated by designing two guide RNAs (Guide RNA 1, TTTTCACACCTCATTTTTTAAGG; Guide RNA 2, TTGAATACTCCACCA Statistical Analysis. All of the experiments were performed at least three ATGCTTGG) using an online CRISPR-P designer tool (http://crispr.hzau.edu. times. For statistical comparisons, we used Student’s t test. Significant dif- cn/CRISPR2/). Then oligos (SI Appendix, Table S1) were designed and cloning ferences are denoted by asterisks. Horizontal lines in all of the graphs was done following a previously described protocol (61). The template for represent SEM. cloning was amplified from pCBC-DT1T2 with the help of oligos. The dele- tions were confirmed by sequencing (SI Appendix, Fig. S5). The PCR frag- Data Availability. This study did not generate any unique datasets or code. All ment was cloned into pHSE401 binary vector by Golden Gate reaction for of the associated protocols and materials used in the paper will be made plant transformation. To generate an AIL1-SRDX construct, nucleotides available to readers upon adequate request. encoding the 12-amino-acid SRDX repressor domain [LDLDLELRLGFA (44)] was introduced into a reverse primer for AIL1, which was used together with ACKNOWLEDGMENTS. This work was supported by grants from Swedish a forward primer to generate nucleotides needed for a full-length AIL1- Foundation for Strategic Research (SSF), the Swedish Research Council for SRDX construct. This PCR product was then cloned into donor vector pENTR/ Environment, Agricultural Sciences and Spatial Planning (FORMAS), and the D-TOPO and subsequently pH2GW7 to generate pH2GW7-AIL1-SRDX. BHK4- Knut and Alice Wallenberg Foundation (2014-0032) (to R.P.B.).

1. M. A. Domagalska, O. Leyser, Signal integration in the control of shoot branching. 14. M. Wang et al., BRANCHED1: A key hub of shoot branching. Front Plant Sci 10,76 Nat. Rev. Mol. Cell Biol. 12, 211–221 (2011). (2019). 2. C. Rameau et al., Multiple pathways regulate shoot branching. Front Plant Sci 5, 741 15. R. K. Singh, T. Svystun, B. AlDahmash, A. M. Jönsson, R. P. Bhalerao, Photoperiod- and (2015). temperature-mediated control of phenology in trees: A molecular perspective. New 3. F. F. Barbier, E. A. Dun, S. C. Kerr, T. G. Chabikwa, C. A. Beveridge, An update on the Phytol. 213, 511–524 (2017). signals controlling shoot branching. Trends Plant Sci. 24, 220–236 (2019). 16. J. P. Maurya, R. P. Bhalerao, Photoperiod- and temperature-mediated control of 4. K. V. Thimann, F. Skoog, Studies on the growth hormone of plants: III. The inhibiting growth cessation and dormancy in trees: A molecular perspective. Ann. Bot. 120, action of the growth substance on bud development. Proc. Natl. Acad. Sci. U.S.A. 19, 351–360 (2017). 714–716 (1933). 17. M. Muhr, N. Prüfer, M. Paulat, T. Teichmann, Knockdown of strigolactone bio- 5. J. M. Horgan, P. F. Wareing, Cytokinins and the growth-responses of seedlings of synthesis genes in Populus affects BRANCHED1 expression and shoot architecture. betula-pendula Roth and acer-pseudoplatanus L to nitrogen and phosphorus de- New Phytol. 212, 613–626 (2016). ficiency. J. Exp. Bot. 31, 525–532 (1980). 18. M. Muhr, M. Paulat, M. Awwanah, M. Brinkkötter, T. Teichmann, CRISPR/Cas9-me- 6. K. Takei, H. Sakakibara, M. Taniguchi, T. Sugiyama, Nitrogen-dependent accumula- diated knockout of Populus BRANCHED1 and BRANCHED2 orthologs reveals a major tion of cytokinins in root and the translocation to leaf: Implication of cytokinin species function in bud outgrowth control. Tree Physiol. 38, 1588–1597 (2018). that induces gene expression of response regulator. Plant Cell Physiol. 42, 19. A. Moreno-Cortés et al., Impact of RAV1-engineering on poplar biomass production: 85–93 (2001). A short-rotation coppice field trial. Biotechnol. Biofuels 10, 110 (2017). 7. O. Leyser, Regulation of shoot branching by auxin. Trends Plant Sci. 8, 541–545 (2003). 20. A. Rohde, R. P. Bhalerao, Plant dormancy in the perennial context. Trends Plant Sci. 8. V. Gomez-Roldan et al., Strigolactone inhibition of shoot branching. Nature 455, 12, 217–223 (2007). 189–194 (2008). 21. Y. Yang et al., The TIE1 transcriptional repressor controls shoot branching by directly 9. M. Umehara et al., Inhibition of shoot branching by new terpenoid plant hormones. repressing BRANCHED1 in Arabidopsis. PLoS Genet. 14, e1007296 (2018). Nature 455, 195–200 (2008). 22. J. A. Aguilar-Martínez, C. Poza-Carrión, P. Cubas, Arabidopsis BRANCHED1 acts as 10. B. J. Ferguson, C. A. Beveridge, Roles for auxin, cytokinin, and strigolactone in reg- an integrator of branching signals within axillary buds. Plant Cell 19,458–472 ulating shoot branching. Plant Physiol. 149, 1929–1944 (2009). (2007). 11. M. G. Mason, J. J. Ross, B. A. Babst, B. N. Wienclaw, C. A. Beveridge, Sugar demand, 23. R. K. Singh et al., A genetic network mediating the control of bud break in hybrid not auxin, is the initial regulator of apical dominance. Proc. Natl. Acad. Sci. U.S.A. 111, aspen. Nat. Commun. 9, 4173 (2018). 6092–6097 (2014). 24. R. Mohamed et al., Populus CEN/TFL1 regulates first onset of flowering, axillary 12. M. de Jong et al., Auxin and strigolactone signaling are required for modulation of identity and dormancy release in Populus. Plant J. 62, 674–688 (2010). Arabidopsis shoot branching by nitrogen supply. Plant Physiol. 166, 384–395 (2014). 25. R. C. Elliott et al., AINTEGUMENTA, an APETALA2-like gene of Arabidopsis with 13. F. F. Barbier, J. E. Lunn, C. A. Beveridge, Ready, steady, go! A sugar hit starts the race pleiotropic roles in ovule development and floral organ growth. Plant Cell 8, 155–168 to shoot branching. Curr. Opin. Plant Biol. 25,39–45 (2015). (1996).

Maurya et al. PNAS Latest Articles | 7of8 Downloaded by guest on September 26, 2021 26. Y. Mizukami, R. L. Fischer, Plant organ size control: AINTEGUMENTA regulates growth 44. K. Hiratsu, K. Matsui, T. Koyama, M. Ohme-Takagi, Dominant repression of target and cell numbers during . Proc. Natl. Acad. Sci. U.S.A. 97, 942–947 genes by chimeric repressors that include the EAR motif, a repression domain, in (2000). Arabidopsis. Plant J. 34, 733–739 (2003). 27. A. Karlberg, L. Bako, R. P. Bhalerao, Short day-mediated cessation of growth requires 45. K. Nieminen et al., Cytokinin signaling regulates cambial development in poplar. Proc. the downregulation of AINTEGUMENTALIKE1 transcription factor in hybrid aspen. Natl. Acad. Sci. U.S.A. 105, 20032–20037 (2008). PLoS Genet. 7, e1002361 (2011). 46. O. Leyser, The control of shoot branching: An example of plant information pro- 28. C. E. Hutchison et al., The Arabidopsis histidine phosphotransfer are re- cessing. Plant Cell Environ. 32, 694–703 (2009). dundant positive regulators of cytokinin signaling. Plant Cell 18, 3073–3087 (2006). 47. B. Wang, S. M. Smith, J. Li, Genetic regulation of shoot architecture. Annu. Rev. Plant 29. S. Shannon, D. R. Meeks-Wagner, A mutation in the Arabidopsis TFL1 gene affects Biol. 69, 437–468 (2018). inflorescence meristem development. Plant Cell 3, 877–892 (1991). 48. N. Braun et al., The pea TCP transcription factor PsBRC1 acts downstream of Strigo- – 30. S. J. Liljegren, C. Gustafson-Brown, A. Pinyopich, G. S. Ditta, M. F. Yanofsky, Interac- lactones to control shoot branching. Plant Physiol. 158, 225 238 (2012). tions among APETALA1, LEAFY, and TERMINAL FLOWER1 specify meristem fate. Plant 49. J. Ni et al., promotes shoot branching in the perennial woody plant Ja- – Cell 11, 1007–1018 (1999). tropha curcas. Plant Cell Physiol. 56, 1655 1666 (2015). 31. J. H. Ahn et al., A divergent external loop confers antagonistic activity on floral 50. Y. L. Gao et al., NtBRC1 suppresses axillary branching in tobacco after decapitation. regulators FT and TFL1. EMBO J. 25, 605–614 (2006). Genet. Mol. Res., 10.4238/gmr15049320 (2016). 32. S. Hanano, K. Goto, Arabidopsis TERMINAL FLOWER1 is involved in the regulation of 51. M. Seale, T. Bennett, O. Leyser, BRC1 expression regulates bud activation potential but is not necessary or sufficient for bud growth inhibition in Arabidopsis. Develop- flowering time and inflorescence development through transcriptional repression. ment 144, 1661 –1673 (2017). Plant Cell 23, 3172–3184 (2011). 52. C. P. Coelho, M. A. Minow, A. Chalfun-Júnior, J. Colasanti, Putative sugarcane FT/TFL1 33. H. Böhlenius et al., CO/FT regulatory module controls timing of flowering and sea- genes delay flowering time and alter reproductive architecture in Arabidopsis. Front sonal growth cessation in trees. Science 312, 1040–1043 (2006). Plant Sci 5, 221 (2014). 34. A. Azeez, P. Miskolczi, S. Tylewicz, R. P. Bhalerao, A tree ortholog of APETALA1 53. M. Kaneko-Suzuki et al., TFL1-Like proteins in rice antagonize rice FT-like in mediates photoperiodic control of seasonal growth. Curr. Biol. 24, 717–724 (2014). inflorescence development by competition for complex formation with 14-3-3 and 35. P. Miskolczi et al., Long-range mobile signals mediate seasonal control of shoot FD. Plant Cell Physiol. 59, 458–468 (2018). growth. Proc. Natl. Acad. Sci. U.S.A. 116, 10852–10857 (2019). 54. T. S. Moraes, M. C. Dornelas, A. P. Martinelli, FT/TFL1: Calibrating plant architecture. 36. C. Li et al., Promoting flowering, lateral shoot outgrowth, leaf development, and Front Plant Sci 10, 97 (2019). flower abscission in tobacco plants overexpressing cotton FLOWERING LOCUS T (FT)- 55. C. Gustafson-Brown, B. Savidge, M. F. Yanofsky, Regulation of the Arabidopsis floral like gene GhFT1. Front Plant Sci 6, 454 (2015). homeotic gene APETALA1. Cell 76, 131–143 (1994). 37. H. Tsuji et al., Hd3a promotes lateral branching in rice. Plant J. 82, 256–266 (2015). 56. K. Kaufmann et al., Orchestration of floral initiation by APETALA1. Science 328,85–89 38. C. Y. Hsu et al., FLOWERING LOCUS T duplication coordinates reproductive and (2010). – vegetative growth in perennial poplar. Proc. Natl. Acad. Sci. U.S.A. 108, 10756 10761 57. Y. Han, C. Zhang, H. Yang, Y. Jiao, Cytokinin pathway mediates APETALA1 function in (2011). the establishment of determinate floral meristems in Arabidopsis. Proc. Natl. Acad. 39. P. B. Brewer, E. A. Dun, B. J. Ferguson, C. Rameau, C. A. Beveridge, Strigolactone acts Sci. U.S.A. 111, 6840–6845 (2014). downstream of auxin to regulate bud outgrowth in pea and Arabidopsis. Plant 58. Y. Burko et al., A role for APETALA1/fruitfull transcription factors in tomato leaf – Physiol. 150, 482 493 (2009). development. Plant Cell 25, 2070–2083 (2013). 40. N. Shinohara, C. Taylor, O. Leyser, Strigolactone can promote or inhibit shoot 59. P. L. H. Rinne, L. K. Paul, C. van der Schoot, Decoupling photo- and thermoperiod by branching by triggering rapid depletion of the auxin efflux protein PIN1 from the projected climate change perturbs bud development, dormancy establishment and plasma membrane. PLoS Biol. 11, e1001474 (2013). in the model tree Populus. BMC Plant Biol. 18, 220 (2018). 41. E. A. Dun, A. de Saint Germain, C. Rameau, C. A. Beveridge, Dynamics of strigolactone 60. M. Karimi, A. Depicker, P. Hilson, Recombinational cloning with plant gateway vec- function and shoot branching responses in Pisum sativum. Mol. Plant 6, 128–140 tors. Plant Physiol. 145, 1144–1154 (2007). (2013). 61. H. L. Xing et al., A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC 42. T. Waldie, H. McCulloch, O. Leyser, Strigolactones and the control of plant develop- Plant Biol. 14, 327 (2014). ment: Lessons from shoot branching. Plant J. 79, 607–622 (2014). 62. S. Tylewicz et al., Dual role of tree activation complex component FD in 43. J. P. Maurya et al., Branching regulator BRC1 mediates photoperiodic control of photoperiodic growth control and adaptive response pathways. Proc. Natl. Acad. Sci. seasonal growth in hybrid aspen. Curr. Biol. 30, 122–126.e2 (2019). U.S.A. 112, 3140–3145 (2015).

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